Stanley, Elise F.

We report a single step, simple, repeatable, rapid and reliable technique for simultaneous immunocytochemical staining with two or more rabbit polyclonal antibodies. This technique, which we have dubbed the ‘Pretty Poly’ method, is based on conjugating the antibodies with commercially available, fluorophore-tagged Staphylococcal protein-A (SP-A). Staining is illustrated at the calyx type presynaptic nerve terminal of the chick ciliary ganglion with antibodies directed against three nerve terminal proteins: neurofilaments of the axonal cytoskeleton, and two secretory vesicle proteins, SV2 and cysteine string protein (CSP). Images were deblurred with an iterative deconvolution protocol. Staining with a single polyclonal antibody was bright and had a resolution approaching light microscope limit. Treatment with two different polyclonal antibodies conjugated with contrasting dye-tagged protein-A resulted in double staining without significant crossover that was fully equivalent to the standard primary/secondary technique. The same single step protocol was used to stain with all three rabbit polyclonal antibodies or to combine the technique with a standard monoclonal primary/secondary antibody stain. Thus, the Pretty Poly protocol is a highly flexible, simple and yet effective staining technique that essentially solves the problem of co-staining with multiple polyclonal rabbit antibodies.

Presynaptic CaV2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive GαO G-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of CaV2.2 with syntaxin 1 and GαO. CaV2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with CaV2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the CaV2.2 puncta, whereas the bulk colocalized with MUNC18-1. GαO, whether in its trimeric or monomeric state, did not coimmunoprecipitate with CaV2.2, MUNC18-1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18-1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with CaV2.2 and GαO, as well as in large clusters remote from the release site with MUNC18-1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.